1. Field of the Invention
The present invention provides nucleotide sequences from coryneform bacteria coding for the pgi gene and a process for increasing metabolic flux through the pentose phosphate cycle by attenuating the pgi gene.
2. Background Information
Nucleotides, vitamins and in particular L-amino acids, very particularly lysine and tryptophan, are used in the foodstuffs industry, in animal nutrition, human medicine and the pharmaceuticals industry.
It is known that these substances are produced by fermentation using strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to its great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up of the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.
The performance characteristics of these microorganisms are improved using methods of mutagenesis, selection and mutant selection. In this manner, strains are obtained which are resistant to antimetabolites or are auxotrophic for regulatorily significant intermediates and produce nucleotides, vitamins or amino acids.
For some years, methods of recombinant DNA technology have also been used to improve strains of Corynebacterium which produce nucleotides, vitamins and L-amino acids.
A typical raw material for the production of these compounds is glucose, which is usually used in the form of starch hydrolysate. Sucrose is also used as a raw material.
On cellular absorption, glucose is phosphorylated with consumption of phosphoenolpyruvate (phosphotransferase system) (Malin and Bourd, Journal of Applied Bacteriology 71, 517-523 (1991)) and is then available to the cell as glucose-6-phosphate. Sucrose is converted into fructose and glucose-6-phosphate by a phosphotransferase system (Shio et al., Agricultural and Biological Chemistry 54, 1513-1519 (1990)) and invertase reaction (Yamamoto et al., Journal of Fermentation Technology 64, 285-291 (1986)).
During glucose catabolism, the enzymes glucose-6-phosphate dehydrogenase (EC 1.1.14.9) and glucose-6-phosphate isomerase (EC 5.3.1.9) compete for the substrate glucose-6-phosphate. The enzyme glucose-6-phosphate isomerase catalyses the first reaction step of the Embden-Meyerhof-Parnas pathway or glycolysis, namely conversion into fructose-6-phosphate. The enzyme glucose-6-phosphate dehydrogenase catalyses the first reaction step of the oxidative portion of the pentose phosphate cycle, namely conversion into 6-phosphogluconolactone.
In the oxidative portion of the pentose phosphate cycle, glucose-6-phosphate is converted into ribulose-5-phosphate, so producing reduction equivalents in the form of NADPH. As the pentose phosphate cycle proceeds further, pentose phosphates, hexose phosphates and triose phosphates are interconverted. Pentose phosphates, such as for example 5-phosphoribosyl-1-pyrophosphate are required, for example, in nucleotide biosynthesis. 5-Phosphoribosyl-1-pyrophosphate is moreover a precursor for aromatic amino acids and the amino acid L-histidine. NADPH acts as a reduction equivalent in numerous anabolic biosyntheses. Four molecules of NADPH are thus consumed for the biosynthesis of one molecule of L-lysine from oxalacetic acid.
The significance of the pentose phosphate cycle to biosynthesis and the production of amino acids, in particular L-lysine, by coryneform bacteria is known and has been the focus of much special interest.
Oishi and Aida (Agricultural and Biological Chemistry 29, 83-89 (1965)) have accordingly reported the xe2x80x9chexose monophosphate shuntxe2x80x9d of Brevibacterium ammoniagenes. Investigations using 13C isotope methods by Ishino et al. (Journal of General and Applied Microbiology 37, 157-165 (1991)) into glucose metabolism during glutamic acid and lysine fermentation indicate a correlation between lysine production and metabolic flux through the pentose phosphate pathway.
The inventors set themselves the object of providing a process for increasing metabolic flux through the pentose phosphate cycle.
Nucleotides, vitamins and in particular L-amino acids, very particularly L-lysine and L-tryptophan, are used in the foodstuffs industry, in animal nutrition, human medicine and the pharmaceuticals industry. There is accordingly general interest in providing improved processes for the production of these products.
The present invention provides an isolated polynucleotide containing a polynucleotide sequence selected from the group
a) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide containing the amino acid sequence of SEQ ID no. 2,
b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID no. 2,
c) polynucleotide which is complementary to the polynucleotides of a) or b), and
d) polynucleotide containing at least 100 successive bases of the polynucleotide sequence of a), b) or c).
The present invention also provides the polynucleotide as claimed in claim 1, wherein it preferably comprises replicable DNA containing:
(i) the nucleotide sequence shown in SEQ ID no. 1 or
(ii) at least one sequence which matches the sequence (i) within the degeneration range of the genetic code, or
(iii) at least one sequence which hybridises with the complementary sequence to sequence (i) or (ii) and optionally
(iv) functionally neutral sense mutations in (i).
The present invention also provides
a polynucleotide as claimed in claim 2, containing the nucleotide sequence as shown in SEQ ID no. 1,
a polynucleotide as claimed in claim 2 which codes for a polypeptide which contains the amino acid sequence as shown in SEQ ID no. 2,
a vector containing the polynucleotide as claimed in claim 1, indent d, in particular pMC1, deposited in E. coli DSM 12969.
and coryneform bacteria acting as host cells which contain the vector as claimed in claim 6.
xe2x80x9cIsolatedxe2x80x9d means separated from its natural environment. xe2x80x9cPolynucleotidexe2x80x9d generally relates to polyribonucleotides and polydeoxyribonucleotides, wherein the RNA or DNA may be unmodified or modified.
xe2x80x9cPolypeptidesxe2x80x9d are taken to mean peptides or proteins which contain two or more amino acids connected by peptide bonds.
The polypeptides according to the invention include the polypeptide according to SEQ ID no. 2, in particular those having the biological activity of glucose-6-phosphate isomerase and also those which are at least 70% identical to the polypeptide according to SEQ ID no. 2, preferably being at least 80% and particularly preferably at least 90% to 95% identical to the polypeptide according to SEQ ID no. 2 and having the stated activity.
This invention furthermore relates to a process for the fermentative production of nucleotides, vitamins and in particular L-amino acids, very particularly lysine and tryptophan, using coryneform bacteria which in particular already produce the stated substances and in which the nucleotide sequences coding for the pgi gene are attenuated, in particular expressed at a low level.
In this connection, the term xe2x80x9cattenuationxe2x80x9d describes the reduction in or switching off of the intracellular activity of one or more enzymes (proteins) in a microorganism, which enzymes are coded by the corresponding DNA, for example by using a weak promoter or a gene or allele which codes for a corresponding enzyme having low activity or inactivates the corresponding enzyme (protein) and optionally by combining these measures.
The microorganisms provided by the present invention are capable of producing nucleotides, vitamins and in particular L-amino acids, very particularly lysine and tryptophan, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. The microorganisms may comprise representatives of the coryneform bacteria in particular of the genus Corynebacterium. Within the genus Corynebacterium, Corynebacterium glutamicum may in particular be mentioned, which is known in specialist circles for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are the known wild type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and mutants or strains produced therefrom which produce nucleotides, vitamins or L-amino acids,
such as for example the 5xe2x80x2-inosinic acid producing strains
Corynebacterium ammoniagenes ATCC15190
Corynebacterium ammoniagenes ATCC15454 and
Corynebacterium glutamicum ATCC14998 or
such as for example the 5xe2x80x2-guanylic acid producing strains
Corynebacterium glutamicum ATCC21171 and
Corynebacterium ammoniagenes ATCC19216 or
such as for example the D-pantothenic acid producing strains
Corynebacterium glutamicum 
ATCC13032/pECM3ilvBNCD, pEKEX2panBC and
Corynebacterium glutamicum ATCC13032/pND-D2 or
such as for example the L-lysine producing strains
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464 and
Corynebacterium glutamicum DSM 5714 or
such as for example the L-tryptophan producing strains
Corynebacterium glutamicum ATCC21850 and
Corynebacterium glutamicum KY9218(pKW9901).
The inventors were successful in isolating the novel pgi gene coding for the enzyme glucose-6-phosphate isomerase (EC 5.3.1.9) from C. glutamicum. 
In order to isolate the pgi gene or also other genes from C. glutamicum, a gene library of this microorganism is first constructed in E. coli. The construction of gene libraries is described in generally known textbooks and manuals. Examples which may be mentioned are the textbook by Winnacker: Gene und Klone, Eine Einfxc3xchrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the manual by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). One very well known gene library is that of E. coli K-12 strain W3110, which was constructed by Kohara et al. (Cell 50, 495-508 (1987)) in xcex-vectors. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was constructed using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Bxc3x6rmann et al. (Molecular Microbiology 6(3), 317-326)) again describe a gene library of C. glutamicum ATCC13032 using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). O""Donohue (The Cloning and Molecular Analysis of Four Common Aromatic Amino Acid Biosynthetic Genes from Corynebacterium glutamicum. Ph.D. Thesis, National University of Ireland, Galway, 1997) describes the cloning of C. glutamicum genes using the xcex Zap Expression system described by Short et al. (Nucleic Acids Research, 16: 7583).
A gene library of C. glutamicum in E. coli may also be produced using plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268). Suitable hosts are in particular those E. coli strains with restriction and recombination defects, such as for example strain DH5xcex1 ((Jeffrey H. Miller: xe2x80x9cA Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteriaxe2x80x9d, Cold Spring Harbor Laboratory Press, 1992).
The gene library is then inserted into an indicator strain by transformation (Hanahan, Journal of Molecular Biology 166, 557-580, 1983) or electroporation (Tauch et.al., 1994, FEMS Microbiological Letters, 123:343-347). The indicator strain is distinguished by having a mutation in the gene in question which causes a detectable phenotype. The E. coli mutant DF1311 described by Kupor and Fraenkel (Journal of Bacteriology 100: 1296-1301 (1969)) is of significance for the purposes of the present invention. This strain carries mutations in the pgi and pgl genes, as a result of which growth on glucose is severely inhibited. After transformation with a vector containing the pgi gene, growth on glucose is re-established. One example of such a vector containing the pgi gene is pAMC1 (FIG. 1).
The long DNA fragments cloned with the assistance of cosmids or other xcex-vectors may subsequently in turn be sub-cloned in usual vectors suitable for DNA sequencing.
DNA sequencing methods are described inter alia in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America USA, 74:5463-5467, 1977).
The resultant DNA sequences may then be investigated using known algorithms or sequence analysis programs, for example Staden""s program (Nucleic Acids Research 14, 217-232(1986)), Butler""s GCG program (Methods of Biochemical Analysis 39, 74-97 (1998)), Pearson and Lipman""s FASTA algorithm (Proceedings of the National Academy of Sciences USA 85,2444-2448 (1988)) or Altschul et al.""s BLAST algorithm (Nature Genetics 6, 119-129 (1994)) and compared with the sequence entries available in publicly accessible databases. Publicly accessible nucleotide sequence databases are, for example, the European Molecular Biology Laboratory database (EMBL, Heidelberg, Germany) or the National Center for Biotechnology Information database (NCBI, Bethesda, Md., USA).
These were the methods used to obtain the novel DNA sequence coding for the pgi gene from C. glutamicum, which is provided by the present invention as SEQ ID no. 1. The amino acid sequence of the corresponding protein was furthermore deduced from the above DNA sequence using the methods described above. SEQ ID no. 2 shows the resultant amino acid sequence of the product of the pgi gene.
Coding DNA sequences arising from SEQ ID NO.1 by the degeneracy of the genetic code are also provided by the present invention. Similarly, DNA sequences which hybridise with SEQ ID no. 1 or portions of SEQ ID no. 1 are also provided by the present invention. Finally, DNA sequences produced by the polymerase chain reaction (PCR) using primers obtained from SEQ ID no. 1 are also provided by the present invention.
The person skilled in the art may find instructions for identifying DNA sequences by means of hybridisation inter alia in the manual xe2x80x9cThe DIG System Users Guide for Filter Hybridizationxe2x80x9d from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). The person skilled in the art may find instructions for amplifying DNA sequences using the polymerase chain reaction (PCR) inter alia in the manual by Gait: Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
The inventors discovered that, after attenuation of the pgi gene, coryneform bacteria exhibit an improved metabolic flux through the pentose phosphate cycle and produce nucleotides, vitamins and in particular L-amino acids, particularly preferably L-lysine and L-tryptophan, in an improved manner.
Attenuation may be achieved by reducing or switching off either the expression of the pgi gene or the catalytic properties of the enzyme protein. Both measures may optionally be combined.
Reduced gene expression may be achieved by appropriate control of the culture or by genetic modification (mutation) of the signal structures for gene expression. Signal structures for gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators. The person skilled in the art will find information in this connection for example in patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al. (Microbiology 142: 1297 (1996) and in known textbooks of genetics and molecular biology, such as for example the textbook by Knippers (xe2x80x9cMolekulare Genetikxe2x80x9d, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or by Winnacker (xe2x80x9cGene und Klonexe2x80x9d, VCH Verlagsgesellschaft, Weinheim, Germany, 1990).
Mutations which result in modification or reduction of the catalytic properties of enzyme proteins are known from the prior art; examples which may be mentioned are the papers by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Mxc3x6ckel (xe2x80x9cDie Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzymsxe2x80x9d, Forschungszentrum Jxc3xclich reports, Jxc3xcl-2906, ISSN09442952, Jxc3xclich, Germany, 1994). Summary presentations may be found in known textbooks of genetics and molecular biology such as, for example, the textbook by Hagemann (xe2x80x9cAllgemeine Genetikxe2x80x9d, Gustav Fischer Verlag, Stuttgart, 1986).
Mutations which may be considered are transitions, insertions, deletions and transversions Depending upon the effect of exchanging the amino acids upon enzyme activity, the mutations are known as missense mutations or nonsense mutations. Insertions or deletions of at least one base pair in a gene give rise to frame shift mutations, as a result of which the incorrect amino acids are inserted or translation terminates prematurely. Deletions of two or more codons typically result in a complete breakdown of enzyme activity. Instructions for producing such mutations belong to the prior art and may be found in known textbooks of genetics and molecular biology, such as for example the textbook by Knippers (xe2x80x9cMolekulare Genetikxe2x80x9d, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), by Winnacker (xe2x80x9cGene und Klonexe2x80x9d, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or by Hagemann (xe2x80x9cAllgemeine Genetikxe2x80x9d, Gustav Fischer Verlag, Stuttgart, 1986).
One example of insertion mutagenesis is plasmid pMC1 (FIG. 2), by means of which the pgi gene may be mutated. Plasmid pMC1 consists of plasmid pBGS8, described by Spratt et al. (Gene 41: 337 (1986)), into which an internal fragment of the pgi gene, shown in SEQ ID no. 3, has been inserted. After transformation and homologous recombination into the pgi gene (insertion), this plasmid brings about a complete loss of enzyme function. Instructions and explanations relating to insertion mutagenesis may be found, for example, in Schwarzer and Pxc3xchler (Bio/Technology 9, 84-87 (1991)) or Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)).
In addition to attenuation of the pgi gene, it may additionally be advantageous for the production of nucleotides, vitamins and in particular L-amino acids, very particularly L-lysine and L-tryptophan, to amplify, in particular overexpress, one or more enzymes of the particular biosynthetic pathway.
For example, when producing nucleotides, it is thus possible
simultaneously to overexpress the purF gene coding for glutamine-PRPP amidotransferase and/or
simultaneously to overexpress the carAB gene coding for carbamoylphosphate synthetase.
For example, when producing L-lysine, it is thus possible
simultaneously to overexpress the dapA gene coding for dihydrodipicolinate synthase (EP-B 0 197 335), and/or
simultaneously to amplify a DNA fragment which imparts S-(2-aminoethyl)cysteine resistance (EP-A 0 088 166).
For example, when producing L-tryptophan, it is thus possible
simultaneously to overexpress the tkt gene coding for transketolase and/or
simultaneously to overexpress the prs gene coding for phosphoribosylpyrophosphate synthase.
Apart from attenuating the pgi gene, it may furthermore be advantageous for the production of nucleotides, vitamins and in particular L-amino acids, very particularly L-lysine and L-tryptophan, to switch off unwanted secondary reactions (Nakayama: xe2x80x9cBreeding of Amino Acid Producing Micro-organismsxe2x80x9d, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
The microorganisms containing the polynucleotide as claimed in claim 1 are also provided by the invention and may be cultured continuously or discontinuously using the batch process or the fed batch process or repeated fed batch process for the purpose of producing nucleotides, vitamins and in particular L-amino acids, very particularly L-lysine and L-tryptophan. A summary of known culture methods is given in the textbook by Chmiel (Bioprozesstechnik 1. Einfxc3xchrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must adequately satisfy the requirements of the particular strains. Culture media for various microorganisms are described in xe2x80x9cManual of Methods for General Bacteriologyxe2x80x9d from American Society for Bacteriology (Washington D.C., USA, 1981). Carbon sources which may be used include sugars and carbohydrates, such as for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as for example soya oil, sunflower oil, peanut oil and coconut oil, fatty acids, such as for example palmitic acid, stearic acid and linoleic acid, alcohols, such as for example glycerol and ethanol, and organic acids, such as for example acetic acid. These substances may be used individually or as a mixture. Nitrogen sources which may be used comprise organic compounds containing nitrogen, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya flour and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture. Phosphorus sources which may be used are phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding salts containing sodium. The culture medium must furthermore contain metal salts, such as for example magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth-promoting substances such as amino acids and vitamins may also be used in addition to the above-stated substances. Suitable precursors may furthermore be added to the culture medium. The stated feed substances may be added to the culture as a single batch or be fed appropriately during cultivation.
Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds, such as phosphoric acid or sulfuric acid, are used appropriately to control the pH of the culture. Antifoaming agents, such as for example fatty acid polyglycol esters, may be used to control foaming. Suitable selectively acting substances, such as for example antibiotics, may be added to the medium in order to maintain plasmid stability. Oxygen or gas mixtures containing oxygen, such as for example air, are introduced into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 20xc2x0 C. to 45xc2x0 C. and preferably from 25xc2x0 C. to 40xc2x0 C. The culture is continued until a maximum quantity of the desired product has been formed. This objective is normally achieved within 10 hours to 160 hours.
Metabolic flux through the pentose phosphate cycle is determined by using a culture containing C-1 labelled 13C glucose as the carbon source. This analytical method is based on the known fact that when glucose is catabolised by the pentose phosphate cycle, the C-1 position is converted into carbon dioxide, whereas when it is catabolised by glycolysis, the 13C-1 labelling is passed on to the C-3 position of the pyruvate. The 13C content of the C-3position of the pyruvate is determined at the appropriate time by using nuclear magnetic resonance or mass spectroscopy methods to investigate extracellular metabolites, such as for example lactate and in particular lysine. Alternatively, amino acids may be obtained by acid hydrolysis from the biomass and the 13C content in the individual carbon atoms of the particular amino acid may then be determined. The person skilled in the art may find comprehensive instructions, in particular in relation to computer-aided data evaluation of the 13C content in various carbon atoms of the investigated metabolites in Sonntag et al. (European Journal of Biochemistry 213, 1325-1331 (1993)), Sonntag et al. (Applied Microbiology and Biotechnology 44, 489-495 (1995)), Marx et al. (Biotechnology and Bioengineering 49, 111-129 (1996)) and Marx et al. (Biotechnology and Bioengineering 56, 168-180 (1997)).
Methods for determining nucleotides, vitamins and L-amino acids are known from the prior art. L-Amino acids may, for example, be analysed using anion exchange chromatography with subsequent ninhydrin derivatisation, as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190), or they may be analysed by reversed phase HPLC as described in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).
The following microorganism has been deposited with Deutschen Sammlung fxc3xcr Mikrorganismen und Zellkulturen (DSMZ, Braunschweig, [sic] The following microorganism has been deposited with Deutschen Sammlung fxc3xcr Mikrorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty:
Escherichia coli strain DH5xcex1/pMC1 as DSM 22969
The following examples will further illustrate this invention. The molecular biology techniques, e.g. plasmid DNA isolation, restriction enzyme treatment, ligations, standard transformations of Escherichia coli etc. used are, (unless stated otherwise), described by Sambrook et al., (Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor Laboratories, USA).
A DNA library of Corynebacterium glutamicum strain ASO19 (Yoshihama et al., Journal of Bacteriology 162, 591-597 (1985)) was constructed using xcex Zap Express(trademark) system, (Short et al., (1988) Nucleic Acids Research, 16: 7583-7600), as described by O""Donohue (O""Donohue, M. (1997). The Cloning and Molecular Analysis of Four Common Aromatic Amino Acid Biosynthetic Genes from Corynebacterium glutamicum. Ph.D. Thesis, National University of Ireland, Galway). xcex Zap Express(trademark) kit was purchased from Stratagene (Stratagene, 11011 North Torrey Pines Rd., La Jolla, Calif. 92037) and used according to the manufacturer""s instructions. AS019-DNA was digested with restriction enzyme Sau3A and ligated to BamHI treated and dephosphorylated xcex Zap Express(trademark) arms.
1. Cloning
Escherichia coli strain DF1311, carrying mutations in the pgi and pgl genes as described by Kupor and Fraenkel, (Journal of Bacteriology 100: 1296-1301 (1969)), was transformed with approx. 500 ng of the AS019 xcex Zap Express(trademark) plasmid library described in Example 1. Selection for transformants was made on M9 minimal media, (Sambrook et al., (1989). Molecular Cloning. A Laboratory Manual Cold Spring Harbor Laboratories, USA), containing kanamycin at a concentration of 50 mg/l and incubation at 37xc2x0 C. for 48 hours. Plasmid DNA was isolated from one transformant according to Birnboim and Doly (Nucleic Acids Research 7: 1513-1523 (1979)) and designated pAMC1 (FIG. 1).
2. Sequencing
For sequence analysis of the cloned insert of pAMC1 the method of Sanger et al. (Proceedings of the National Academy of Sciences USA 74, 5463-5467 (1977)) was applied using primers differentially labelled with a coloured fluorescent tag. It was carried out using the ABI prism 310 genetic analyser from Perkin Elmer Applied Biosystems, (Perkin Elmer Corporation, Norwalk, Conn., U.S.A), and the ABI prism Big Dye(trademark) Terminator Cycle Sequencing Ready Reaction kit also from Perkin Elmer.
Initial sequence analysis was carried out using the universal forward and M13 reverse primers obtained from Pharnacia Biotech (St. Albans, Herts, AL1 3AW UK):
Universal forward primer: GTA ATA CGA CTC ACT ATA GGG C (SEQ ID NO:4)
M13 reverse primer: GGA AAC AGC TAT GAC CAT G (SEQ ID NO:5) Internal primers were subsequently designed from the sequence obtained which allowed the entire pgi gene to be deduced. The sequence of the internal primers is as follows:
Internal primer 1: GGA AAC AGG GGA GCC GTC (SEQ ID NO:6)
Internal primer 2: TGC TGA GAT ACC AGC GGT (SEQ ID NO:7)
Sequence obtained was then analysed using the DNA Strider program (Marck, (1988)). Nucleic Acids Research 16: 1829-1836), version 1.0 on an Apple Macintosh computer. This program allowed for analyses such as restriction site usage, open reading frame analysis and codon usage determination. Searches between DNA sequence obtained and those in EMBL and Genbank databases were achieved using the BLAST program, (Altschul et al., (1997). Nucleic Acids Research, 25: 3389-3402). DNA and protein sequences were aligned using the Clustal V and Clustal W programs (Higgins and Sharp, 1988 Gene 73: 237-244).
The sequence thus obtained is shown in SEQ ID NO 1. The analysis of the nucleotide sequence obtained revealed an open reading frame of 1650 base pairs which was designated as pgi gene. It codes for a protein of 550 amino acids shown in SEQ ID NO 2.
1. Construction of a pgi disruption vector
An internal segment of the pgi gene was amplified by polymerase chain reaction (PCR) using genomic DNA isolated from Corynebacterium glutamicum ASO19, (Heery and Dunican, (1993) Applied and Environmental Microbiology 59: 791-799), as template. The pgi primers used were:
fwd. Primer: ATG GAR WCC AAY GGH AA (SEQ ID NO:8)
rev. Primer: YTC CAC GCC CCA YTG RTC (SEQ ID NO:9)
with R=A+G; Y=C+T; W=A+T; H=A+T+C.
PCR Parameters were as follows:
35 cycles
94xc2x0 C. for 1 min.
47xc2x0 C. for 1 min.
72xc2x0 C. for 30 sec.
1.5 mM MgCl2 
approx. 150-200 ng DNA template.
The PCR product obtained was cloned into the commercially available PGEM-T vector received from Promega Corp., (Promega UK, Southampton) using strain E. coli JM109, (Yanisch-Perron et al., 1985. Gene, 33: 103-119), as a host. The sequence of the PCR product is shown as SEQ ID No. 3. The cloned insert was then excised as an EcoRI fragment and ligated to plasmid pBGS8 (Spratt et al., Gene 41: 337-342 (1986)) pretreated with EcoRI. The restriction enzymes used were obtained from Boehringer Mannheim UK Ltd., (Bell Lane, Lewes, East Sussex BN7 1LG, UK) and used according to the manufacturer""s instructions. E. coli JM109 was then transformed with this ligation mixture and electrotransformants were selected on Luria agar supplemented with IPTG (isopropyl-xcex2-D-thiogalactopyranoside), XGAL (5-bromo-4-chloro-3-indolyl-D-galactopyranoside) and kanamycin at a concentration of 1 mM, 0.02% and 50 mg/l respectively. Agar plates were incubated for twelve hours at 37xc2x0 C. Plasmid DNA was isolated from one transformant, characterised by restriction enzyme analysis using EcoRI, BamHI and SalI designated pMC1 (FIG. 2).
2. Insertion Mutagenesis of the pgi Gene in Strain DSM5715
Strain DSM 5715 was then transformed with plasmid pMC1 using the electroporation method described by Liebl et al. (FEMS Microbiology Letters, 53:299-303 (1989)).
Transformant selection proceeded on LBHIS agar consisting of 18.5 g/l of brain-heart infusion bouillon, 0.5 M sorbitol, 5 g/l of Bacto tryptone, 2.5 g/l of Bacto yeast extract, 5 g/l of NaCl and 18 g/l of Bacto agar, which had been supplemented with 15 mg/l of kanamycin and 1% fructose. Incubation was performed for 2 days at 33xc2x0 C. Transformants 1, 2 and 3 were obtained.
The resultant transformants were were tested using the polymerase chain reaction (PCR). To this end, chromosomal DNA was isolated from the transformants obtained and from strain DSM5715 as described in Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). The following primer oligonucleotides were selected for PCR on the basis of the DNA sequence of the pgi gene, which is shown in SEQ ID NO:1:
pgi-1: 5xe2x80x2 ACC CAC GCT GTC CTA CCT TA 3xe2x80x2 (SEQ ID NO:10)
pgi-2: 5xe2x80x2 TGT CCC AAA TCA CGC CCT AG 3xe2x80x2 (SEQ ID NO:11)
pgi-3: 5xe2x80x2 gat gat agc ggc cag tgc at 3xe2x80x2 (SEQ ID NO:12).
The primers shown were synthesised by the company MWG Biotech (Ebersberg, Germany) and the PCR reaction performed using the standard PCR method of Innis et al. (PCR-Protocols. A guide to methods and applications, 1990, Academic Press). The chromosomal DNA of the transformants was used as the template, and the chromosomal DNA of DSM5715 was used as the control. Each template was used in two PCR reactions, one with the primer pair pgi-1/pgi-2 and one with the primer pair pgi-1/pgi-3.
The PCR batches were separated by electrophoresis in a 0.8% agarose gel. Using primer pair pgi-1/pgi-2, each of the four PCR reactions yielded a DNA fragment of a length of 0.5 kb. Using primer pair pgi-1/pgi-3, only the control with DSM5715 DNA showed an amplification product of a length of 0.7 kb. No PCR product could be detected in the batches with chromosomal DNA from the transformants.
Transformant no. 3 characterised in this manner was named strain DSM5715:pMC1.